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Eight large-scale RNAi libraries designed for the Drosophila genome (Ambion, HD2, HFA/DRSC v1.0, DRSC v2.0, OpenBiosystems v1/v2, MRC, NIGFLY and VDRC) were re-annotated using the FlyBase annotations of release 5.24.



NEXT-RNAi results

HTML reports

NEXT-RNAi HTML ouputs for the library evaluations are available here:

Pairwise comparisons of libraries

We used NEXT-RNAi results to identify the number of genes that could be targeted with independent designs through pairwise combinations of all Drosophila RNAi libraries. A summary report for all pairwise combinations is contained in libComparison_report.txt (44KB).

A detailed analysis for each combination of two libraries mapping reagents available for a certain target gene in one library to the reagents for this gene available in the other library (including information whether these reagents are 'completely', 'partially' or 'not' independent ) is available for download (libComparison.tar.gz (15MB).


Input files and settings used

Input FASTA files

Ambion, DRSC v2.0 and HD2 libraries were evaluated using long dsRNA sequences as input; HFA/DRSC v1.0, OpenBiosystems v1/v2, MRC and VDRC libraries were evaluated using both, primer and long dsRNA sequences as input and the NIGFLY library was evaluated starting from primers.

Targetgroup file (tab-delimited)

FBtr-FBgn-CG-Txn-Name-r5.24.tab (1.4MB) defining which FlyBase transcripts belong to the same gene (headers Target and TargetGroup) (TARGETGROUPS option)

Target	        TargetGroup	#1	        #2	        #3	#4
FBtr0085315	FBgn0039596	CG10000-RA	CG10000-RA	CG10000	CG10000
FBtr0085316	FBgn0039595	AR-2-RA	        CG10001-RA	CG10001	AR-2
FBtr0085321	FBgn0000659	fkh-RA    	CG10002-RA	CG10002	fkh
FBtr0300259	FBgn0000659	fkh-RB	        CG10002-RB	CG10002	fkh
FBtr0082507	FBgn0037972	CG10005-RA	CG10005-RA	CG10005	CG10005

Bowtie database/index for off-target evaluation

Bowtie database/index containing annotated FlyBase (release 5.24) transcripts for specificity calculations (-d input):
dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta.tar.gz (55MB)

Bowtie database/index for mapping of reagents

The Bowtie database/index (GENOMEBOWTIE option) for mapping of reagents to the Drosophila genome:
dmel-all-chromosome-r5.24.tar.gz (154MB)
Bowtie indexes for genomes are also available through the Bowtie webpage.

Feature file with UTR locations

Tab-delimited feature file containing FlyBase mappings of UTRs to chromosomes that is used to calculate the 'UTR-content' (FEATURE option) of designed reagents: dmel-all-UTRs-r5.24.zip (65MB)

UTRID    FeatureName    FeatureLoc    FeatureStart    FeatureEnd
UTR_1    UTR            2RHet         2901830         2901830
UTR_2    UTR            2RHet         1335191         1335191
UTR_3    UTR            2RHet         198854          198854

FASTA file for homology evaluation

Transcriptome FASTA file to evaluate the homology of the designs using Blast (HOMOLOGY option):
dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta.zip (14MB)

FASTA file containing miRNAs (miRBase)

FASTA file containing miRNAs downloaded from miRBase used to avoid conserved miRNA seeds from long dsRNA designs (MIRSEED option):
miRBase_r14_Dmel.fa (16KB)


Evaluation criteria

Start of program

Ambion

perl nextrnai.pl -i ambion_amplicon.fa -s 14000 -r d
-d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA
-o options.txt -n Dmelr524_AMB

HD2

perl nextrnai.pl -i HD2Designs_Batch123.fasta -s 14000 -r d
-d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA
-o options.txt -n Dmelr524_HD2

HFA

perl nextrnai.pl -i HFA_dsRNA.fasta+HFA_primer.fasta -s 14000 -r d
-d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta -e DSRNA+OLIGO
-o options.txt -n Dmelr524_HFA

DRSC v2.0

perl nextrnai.pl -i v2_seqs_MOD.fa -s 14000 -r d
-d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta
-e DSRNA -o options.txt -n Dmelr524_DRSCv2

OpenBiosystems v1/v2

perl nextrnai.pl -i ob_v01_v02_dsRNA.fasta+ob_v01_v02_primer.fasta -s 14000 -r d
-d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta
-e DSRNA+OLIGO -o options.txt -n Dmelr524_OBS_v01_v02

MRC

perl nextrnai.pl -i mrc_amplicon.fa+mrc_primer.fa -s 14000 -r d
-d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta
-e DSRNA+OLIGO -o options.txt -n Dmelr524_MRC

NIGFLY

perl nextrnai.pl -i nigfly_primer.fa -s 15000 -r d
-d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta
-e OLIGO -o options.txt -n Dmelr524_NIGFLY

VDRC

perl nextrnai.pl -i vdrc_amplicon.fa+vdrc_primer.fa -s 14000 -r d
-d dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta
-e DSRNA+OLIGO -o options.txt -n Dmelr524_VDRC


Descriptions for start parameters used are available here.

Options file

The same options file was used for all evaluation runs.


DESIGNWINDOW=1,10000
SIRNALENGTH=19
EFFICIENCY=SIR,63
BOWTIE=/usr/bin/
TARGETGROUPS=FBtr-FBgn-CG-Txn-Name-r5.24.tab
PRIMER3=/usr/bin/
GENOMEBOWTIE=dmel-all-chromosome-r5.24
BLAT=/usr/bin/
BLATPROGRAM=gfClient
BLATHOST=b110-cellarray3
BLATPORT=3501
GENOMEFASTA=dmel-all-chromosome-r5.24.fasta
GFF=GFF3
GBROWSEBASE=http://www.dkfz.de/signaling/cgi-bin/gbrowse_img/flybase/
GBROWSETRACK=GENE+TXN
AFF=YES
LOWCOMPEVAL=/usr/bin/
CANEVAL=6
HOMOLOGY=/usr/bin/,dmel-all-txn-miRNA-miscRNA-ncRNA-pseudogene-tRNA-r5.24.fasta,1e-10
FEATURE=dmel-all-UTRs-r5.24.tab
MIRSEED=7,miRBase_r14_Dmel.fa


Descriptions for all options used are available here.


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